Permselective membrane for treating vascular calcifications in chronic hemodialysis patients

ABSTRACT

The present disclosure relates to a hemodialysis membrane for the treatment of vascular calcification in hemodialysis patients, especially in chronic hemodialysis patients. The present disclosure further relates to methods of treating vascular calcification in hemodialysis patients, wherein the hemodialysis membrane is characterized in that it comprises at least one hydrophobic polymer and at least one hydrophilic polymer and in that it has a MWRO of between 15 and 20 kD and a MWCO of between 170-320 kD or that the hemodialysis membrane comprises at least one hydrophobic polymer and at least one hydrophilic polymer and has a MWRO of between 8.5 kD and 14.0 kD and a MWCO of between 55 kD and 130 kD.

TECHNICAL FIELD

The present disclosure relates to a hemodialysis membrane for thetreatment of vascular calcification in hemodialysis patients, especiallyin chronic hemodialysis patients. The present disclosure further relatesto methods of treating vascular calcification in hemodialysis patients,wherein the hemodialysis membrane is characterized in that it comprisesat least one hydrophobic polymer and at least one hydrophilic polymerand in that it has a MWRO of between 15 and 20 kD and a MWCO of between170-320 kD, or, in the alternative, has a MWRO of between 8.5 and 14 kDand a MWCO of between 55 kD and 130 kD.

DESCRIPTION OF THE RELATED ART

Patients with impaired renal function due to chronic kidney diseasesface one of the highest risks for cardiovascular morbidity and deaththat continuously increases as kidney function declines. This is truefor patients with pre-end-stage renal failure, on dialysis or aftersuccessful renal transplantation. It is the most common cause for deathin patients with a functional allograft, and prevents many dialysispatients from being engrafted (Goldsmith et al. (2001): “Coronary arterydisease in patients with renal failure”, Int J Clin Pract 55, 196-210).The prevailing metabolic milieu in moderate-to-severe chronic renalfailure and on dialysis strongly seems to favor an increased rate ofatherosclerosis and atherosclerotic/thrombotic events in these patients.There is now evidence that vascular smooth muscle cells can becomechondrocyte or osteoblast-like and lay down and mineralize collagen andnon-collagenous proteins in arteries. Resulting vascular calcificationremains one of the major unsolved problems in uremic patients. Arterialcalcium load seems to be a strong predictor for cardiovascularcomplications in this population.

Vascular calcification is common in physiologic and pathologicconditions such as aging, diabetes, dyslipidemia, genetic diseases, anddiseases with disturbances of calcium metabolism. However, in CKDpatients, vascular calcification is even more common, developing earlyand contributing to the markedly increased cardiovascular risk.Increased knowledge about the mechanisms of calcification together withimproved imaging techniques have provided evidence that vascularcalcification should be divided into two distinct entities according tothe specific site of calcification within the vascular wall. Intimalcalcification is advanced atherosclerosis, occurring in medium-to-largeconduit arteries without smooth muscle cells. Plaques develop andarterial occlusion occurs. Medial calcification, known also asMönckeberg's arteriosclerosis, occurs in elastin fibers around smoothmuscle cells in the absence of atherosclerosis or inflammation and isseen primarily in chronic renal failure or diabetes. It is typicallyless occlusive of the arterial lumen than intimal calcification.(Nakamura et al (2009): “Coronary Calcification in Patients with chronickidney disease and coronary artery disease”. Clin J Am Soc Nephrol 4,1892-1900). Also, medial calcification occurs in arteries of any size,including small arteries in which atherosclerosis does not occur. Renalfailure increases the extent of calcification in atheroscleroticplaques, but the effect on medial calcification is probably greater asit rarely occurs in individuals without renal insufficiency under theage of 60 years. The histological prevalence of medial calcification inradial arteries was 45-fold greater in patients with CKD compared withthose without CKD (O'Neill et al. (2010): “Recent progress in thetreatment of vascular calcification”. Kidney Int. 78(12), 1232-1239).

In general, the presence of vascular calcifications in end-stage renaldisease (ESRD) patients is associated with increased stiffness of large,capacitive, elastic-type arteries like the aorta and common carotidartery (CCA) (Blacher et al. (2001): “Arterial Calcifications, ArterialStiffness, and Cardiovascular Risk in End-Stage Renal Disease”.Hypertension 38, 938-942), together with higher pulse wave velocity(PWV), earlier return of wave reflections from the periphery to theascending aorta during systole and abnormal increase of aortic systolicblood pressure, with reduced diastolic blood pressure and high pulsepressure. In the general population and in patients with CKD,electron-beam computed tomography (EBCT) has proven coronary arterycalcification (CAC) as a potent predictor of cardiac events. Both theprevalence and intensity of CAC are increased in patients with CKD.

Traditional cardiac risk factors do not appear to entirely account forthe elevated cardiovascular morbidity seen in advanced CKD.Hyperphosphatemia, elevated Ca×P product, and hyperparathyroidism havebeen associated with cardiovascular disease (CVD) risk and mortality inadvanced CKD. In addition, uremia is believed to confer nontraditionalCVD risks such as, for example, a proinflammatory state anddysregulation of calcification inhibitors and inducers.

Calcification in patients with end-stage renal disease (ESRD) waspreviously believed to occur as a result of passive mineral depositionprocesses. Although the pathophysiology is not completely understood, itis clear that it is a multifactorial process involving altered mineralmetabolism, as well as changes in systemic and local factors that canpromote or inhibit vascular calcification, and all of these arepotential therapeutic targets. Molecular Mechanisms involved in vascularcalcifaction are described, for example, in Mizobuchi et al. (2009):“Vascular Calcification: The Killer of Patients with Chronic KidneyDisease”, J Am Soc Nephrol 20, 1454, namely ectopic osteogenesis andelastin degradation. Inducers of vascular calcification are alsoreviewed in Mizobuchi et al. (2009): “Vascular Calcification: The Killerof Patients with Chronic Kidney Disease”, J Am Soc Nephrol 20,1453-1464, where it is stated that compared with the general population,patients with CKD have a disproportionately high occurrence of vascularcalcification. One hypothesis to account for this is the altered Ca²⁺and P²⁻ metabolism seen in these patients. This is the most importantcontributor to the progression of vascular calcification in the uremiccondition. Another factor mentioned are uremic toxins. Uremic serum wasfound to upregulate the expression of, for example, Cbfa1/Runx2 and itstarget protein OPN, and increases secretion of a mediator ofosteoblastic differentiation, BMP-2, resulting in the mineralization ofVSMCs into osteoblast-like cells, see also Moe et al. (2008), FIG. 1.Mizobuchi et al. also mention oxidative stress and inflammation andother inducers such as leptin. The bone proteins osteonectin,osteopontin, bone sialoprotein, type I collagen, and alkalinephosphatase have also been identified in multiple sites of extraskeletalcalcification. Interestingly, in cell culture, vascular smooth musclecells and vascular pericytes are capable of producing these sameboneforming transcription factors and proteins, and can be induced to doso with high concentrations of phosphorus, uremic serum, high glucose,oxidized lipids, and several other factors (Moe et al. (2008):“Mechanisms of Vascular Calcification in Chronic Kidney Disease”, J AmSoc Nephrol 19, 213-216.

Therefore, current therapeutic approaches are directed to preventingdisordered bone and mineral metabolism in advanced kidney disease andmainly involve lowering the circulating levels of both phosphate andcalcium. The efficacy of compounds that specifically targetcalcification, such as bisphosphonates and thiosulfate, has been shownin animals but only in small numbers of humans, and safety remains anissue (O'Neill et al. (2010): “Recent progress in the treatment ofvascular calcification”. Kidney Int. 78(12), 1232-1239). Additionaltherapies, such as pyrophosphate, vitamin K, and lowering of pH, aresupported by animal studies, but are yet to be investigated in furtherdetail (O'Neill et al. (2010)). In any case, potential anticalcificationtherapies always carry the risk of adversely acting on normalcalcification, for example in bones and teeth.

Interestingly, not all dialysis patients seem to develop arterialcalcifications, despite similar exposure to these risk factors, andimportantly, do not develop calcification with increased duration ofdialysis (Moe et al. (2008)). For the efficient treatment of CKDpatients, it is therefore helpful to which patients have a high risk fora cardiovascular event. Patients with calcification having a higher riskfor future coronary events than an age-gender-specific percentileranking can then be treated with available therapies. The Kidney DiseaseImproving Global Outcomes (KDIGO) suggests (see Kidney International(2009) (Suppl 113), S1-S2) that patients with CKD stages 3-5 with knownvascular/valvular calcification should generally be considered athighest cardiovascular risk. Coronary artery calcification score (CACS)may be used as a quantitative assessment of calcified atherosclerosiswhich is detectable by electron-beam (EBCT) or multislice computedtomography (CT). The score which is also referred to as Agatston scoreis calculated using a weighed value assigned to the highest density ofcalcification in a given coronary artery. Details on how the Agatstonscore as used herein can be determined and analyzed are given inHalliburton et al. (2010): “Noninvasive quantification of coronaryartery calcification: methods and prognostic value”, Cleve Clin J Med.69 (suppl. 3), S6 -S11. A more elaborate method to determine theAgatston score of a patient is shown in van der Bijl et al. (2010), AJR195, 1299-1305. An Agatston score of 0 is normal. In general, the higherthe score, the more likely it is to have a coronary heart disease (CHD).A score of 0 to 10 is associated to a low risk, a score of 11 to 100 toan intermediate risk. A score of more than 100 (>100) describes anintermediate to high risk. A score of more than 400 (>400) describespersons with a very high risk (Halliburton et al. (2010)).

So far, current therapeutic approaches are, apart from nutritionalaspects, based mainly on a certain medication of the patients in need ofa treatment. The dialysis treatment, in contrast, has mainly beendiscussed with regard to the risks it imposes on a CKD patient and thedevelopment of vascular calcification. However, because of the risks anddrawbacks associated with the compound based anticalcification therapiesdirected to preventing disordered bone and mineral metabolism inadvanced kidney disease, it would clearly be desirable to devise adialysis system which is able to lower or reduce calcification in CKDpatients already during the extracorporeal treatment and before vascularcalcification develops. If a system can be devised which is able toreduce the onset of calcification in CKD patients or reduces existingcalcification in patients who already have to undergo medication, saidmedication and potential side effects related thereto could be reducedor omitted completely.

Currently available membranes and filters for use in hemodialysis,hemodiafiltration or hemofiltration could so far not contributeeffectively to avoiding or reducing vascular calcification in uremicpatients. For the avoidance of doubt, if not expressly indicatedotherwise, the expression “hemodialysis” as used herein encompasseshemodialysis, hemodiafiltration and hemofiltration methods. Based on thefindings on molecular mechanisms involved in vascular calcification asdescribed above and reviewed, for example, in Mizobuchi et al. (2009),the present inventors have focused their attention on the key mediatorsfor the mineralization of cells in the vessel wall and on methods forthe removal of such mediators rather than addressing calcificationproblems by administering inhibitors of such mediators. As a result oftheir studies, the inventors have found that newly developed membranes,so-called high cut-off membranes can be used for eliminating from CKDpatients in need said pro-calcifying mediators which induce and/orpromote calcification. It was found as a consequence that calcificationcan be reduced or delayed by using said high cut-off membranes in theextracorporeal hemodialysis treatment of uremic patients.

In general, dialysis membranes are designed to accomplish the removal ofuremic toxins and excess water from the blood of patients with chronicrenal failure while balancing the electrolyte content in the blood withthe dialysis fluid. Uremic toxins are usually classified according totheir size and physicochemical characteristics in small water-solublecompounds (e.g., urea and creatinine), protein-bound solutes (e.g.,p-cresyl sulfate) and middle molecules (e.g., b2-microglobulin andinterleukin-6) (1-4). While the removal of small molecules takes placemainly by diffusion due to concentration differences between the bloodstream and the dialysis fluid flow, the removal of middle molecules ismainly achieved by convection through ultrafiltration. The degree ofdiffusion and convection depends on the treatment mode (hemodialysis,hemofiltration or hemodiafiltration) as well as on the membrane type(low-flux, high-flux, protein leaking, or high cut-off membranes). Thesieving property of a membrane, i.e., its permeability to solutes, isdetermined by the pore size and sets the maximum size for the solutesthat can be dragged through the membrane with the fluid flow. Thesieving coefficient for a given substance could be simply described asthe ratio between the substance concentration in the filtrate and itsconcentration in the feed (i.e., the blood or plasma), and is thereforea value between 0 and 1. Assuming that the size of a solute isproportional to its molecular weight, a common way to illustrate theproperties of membranes is by creating a sieving curve, which depictsthe sieving coefficient as a function of the molecular weight. Themolecular weight cut-off (MWCO) is defined as the molecular weight wherethe sieving coefficient is 0.1 (FIG. 1). The sieving curve determinedfor a polydisperse dextran mixture can be considered a standardcharacterization technique for a membrane. Conventional dialysismembranes are classified as low-flux or high-flux, depending on theirpermeability. A third group, called protein leaking membranes, is alsoavailable on some markets. These three membrane groups were described ina review by Ward (2005), J Am Soc Nephrol 16, 2421-2430. Lately a fourthtype has emerged, the above-mentioned high cut-off membranes, which haveparticular characteristics (Boschetti-de-Fierro et al. (2013): “Extendedcharacterization of a new class of membranes for blood purification: Thehigh cut-off membranes”, Int J Artif Organs 36(7), 455-463). A concisesummary of the general classification and performance of said membranesas is shown in Table I of Boschetti-de-Fierro et al. (2013) and shall bevalid also for describing the present invention. The latest step inmembrane development is a membrane type which in terms of classificationcould be positioned in between the so-called high flux and the highcut-off membranes. Said membranes are therefore also referred to as“medium cut-off” membranes (see also Table II). These membranes and howthey can be prepared are described in detail in European PatentApplication No. 14154175.5.

TABLE I General classification and typical performance of hemodialysismembranes Sieveing Coefficient^(b) Album Water β2- FLC in Dialyzerpermeability^(a) Micro- Clearance^(c) Loss type ml/(m²hmmHg) globulinAlbumin Kappa Lambda (g)^(d) Low- 10-20 — <0.01 — — 0 flux High- 200-4000.7-0.8 <0.01 <10 <2 <0.5 flux Protein  50-500 0.9-1.0 0.02-0.03 — — 2-6leaking High 1100 1.0 0.2  38 33 28 cut- off ^(a)with 0.9 wt.-% sodiumchloride at 37 ± 1° C. and Q_(B) 100-500 ml/min ^(b)according to EN1283with Q_(B) max and UF 20% ^(c)Serum Free Light Chains, Clearance invitro, Q_(B) 250 ml/min and Q_(D) 500 ml/min, UF 0 ml/min, BovinePlasma, 60 g/l, 37° C., Plasma Level: human κ 500 mg/l, human λ 250mg/l. All clearances in ml/min, measured for membrane areas between 1.7and 2.1 m² ^(d)measured in conventional hemodialysis, after a 4-hsession, with Q_(B) 250 ml/min and Q_(D) 500 ml/min, for membrane areasbetween 1.7 and 2.1 m²

The most evident difference among the types of membranes is theirposition along the molecular weight axis (FIG. 2). High-flux membraneshave a sieving curve more similar to that of the glomerular membrane,removing toxins of small molecular weight such as urea and also allowingsome removal of relatively large toxins, such as β2-microglobulin andmyoglobin. High cut-off membranes show a sieving curve located at highermolecular weights than that for the glomerular membrane. Although thehigh cut-off sieving profile resembles that of the glomerular membraneup to 20 kDa, the high cut-off membranes are open toward molecularweights higher than 20 kDa. This means that the high cut-off membranesallow some passage of proteins. WO 2004/056460 already discloses certainearly high cut-off membranes which could be used for the treatment ofsepsis in dialyzers by eliminating sepsis-associated inflammatorymediators. Advanced dialyzers with high cut-off membranes which arecurrently on the market are, for example, HCO1100®, septeX™ andTheralite®, all available from Gambro Lundia AB. Known uses of highcut-off membranes include treatment of sepsis, chronic inflammation (EP2 161 072 A1), amyloidosis and rhabdomyolysis and treatment of anemia(US 2012/0305487 A1), the most explored therapy to date being thetreatment of myeloma kidney (US 7,875,183 B2). In this case, the removalof the free light chains in patients with multiple myeloma onchemotherapy has allowed the recovery of kidney function in asignificant number of patients. Due to the loss of up to 40 g of albuminper session with the above-mentioned dialyzers, high cut-off membraneshave been used for acute applications, although some physicians havecontemplated benefits of using them in chronic applications, possibly inconjunction with albumin substitution.

The expression “molecular weight cut-off” or “MWCO” or “nominalmolecular weight cut-off” as used herein is a value for describing theretention capabilities of a membrane and refers to the molecular mass ofa solute where the membranes have a rejection of 90% (see above and FIG.1), corresponding to a sieving coefficient of 0.1. The MWCO canalternatively be described as the molecular mass of a solute, such as,for example, dextrans or proteins where the membranes allow passage of10% of the molecules. The shape of the curve depends, for example, onthe pore size distribution and is thus linked to the physical form ofappearance of the membrane.

As already mentioned, sieving curves give relevant information in twodimensions: the shape of the curve describes the pore size distribution,while its position on the molecular weight axis indicates the size ofthe pores. Molecular weight cut-off (MWCO) limits the analysis of thesieving curve to only one dimension, namely to the size of the poreswhere the sieving coefficient is 0.1. To enhance membranecharacterization the molecular weight retention onset (MWRO) is usedherein for characterizing high cut-off and medium cut-off membranes. TheMWRO is defined as the molecular weight at which the sieving coefficientis 0.9, as schematically shown in FIG. 1. It is analogous to the MWCOand describes when the sieving coefficient starts to fall from 1 to 0.Defining two points on the sieving curves allows a bettercharacterization of the sigmoid curve, giving an indication of the poresizes and also of the pore size distribution. The expression “molecularweight rejection onset” or “MWRO” or “nominal molecular weight rejectiononset”, as used herein, therefore refers to the molecular mass of asolute where the membranes have a rejection of 10%, or, in other words,allow passage of 90% of the solute, corresponding to a sievingcoefficient of 0.9.

The use of dextran sieving curves together with the respective MWCO andMWRO values based thereon allows differentiating the existing dialyzertypes low-flux, high-flux, protein leaking, medium cut-off or highcut-off (FIG. 3). Compared to the high-flux dialyzers, which are thestandard for current dialysis treatment, the low-flux dialyzers aredepicted in a group with low MWRO and MWCO. The other twofamilies—protein leaking and high cut-off dialyzers—have differentcharacteristics. While the protein leaking dialyzers are mainlycharacterized by a high MWCO and a low MWRO, the high cut-off family canbe strongly differentiated due to the high in vitro values for both MWROand MWCO (Table II).

TABLE II General classification of hemodialysis membranes based ondextran sieving Structural Characteristics Dialyzer MWRO MWCO Poreradius type [kDa] [kDa] [nm] Low-flux 2-4 10-20 2-3 High-flux  5-1025-65 3.5-5.5 Protein 2-4 60-70 5-6 leaking High cut-off 15-20 170-320 8-12 Medium cut-  8.5-14.0  55-130 5.5 < pore off radius < 8.0

The applicants have found that high cut-off membranes as defined aboveand in Table II as well as medium cut-off membranes as defined above anddescribed in further detail in EP 14154175.5 can be used to effectivelytreat vascular calcification in chronic hemodialysis patients.Especially the high permeability of the high cut-off membranes but alsothe characteristics of the medium cut-off membranes seem to allow for anincreased clearance of relevant mediators in comparison to the high-fluxdialyzers which currently are the standard for treating chronichemodialysis patients. More specifically, even though the uremic milieuis characterized by a multitude of known and so far unidentifiedsubstances, the inventors were able to show a reduction of mediators inthe serum of patients having been treated with high cut-off or mediumcut-off membranes, which serum otherwise could be shown to induceosteoblastic phenotype and osteoblastic differentiation in mesenchymalstem cells (MSC).

SUMMARY OF THE INVENTION

It is the object of the present invention to provide for a method oftreating vascular calcification in a hemodialysis patient, comprisingwithdrawing and bypassing the blood from the patient in a continuousflow into contact with one face of an hemodialysis membrane,simultaneously passing dialysate solution in a continuous flow on anopposite face of the hemodialysis membrane to the side of thehemodialysis membrane in contact with the blood, the flow of thedialysate solution being countercurrent to the direction of flow ofblood, and returning the blood into the patient, wherein thehemodialysis membrane is characterized in that it comprises at least onehydrophobic polymer and at least one hydrophilic polymer and in that ithas a MWRO of between 15 and 20 kD and a MWCO of between 170-320 kD orin that it has a MWRO of between 8.5 kD and 14.0 kD and a MWCO ofbetween 55 and 130 kD. The MWRO and MWCO for a given membrane is basedon dextran sieving experiments before blood contact of the membrane asdescribed by Boschetti-de-Fierro et al. (2013)(see “Materials andMethods” section of the reference).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a dextran sieving curve where the valuesof molecular weight retention onset (MWRO, achieved at SC=0.9) andmolecular weight cut-off (MWCO, achieved at SC=0.1) are illustrated.

FIG. 2 shows characteristic dextran sieving curves for the differenttypes of dialysis membranes: low flux, high flux and high cut-off. Thedata for the glomerular membrane (as reported by Axelsson et al. (2009):Loss of size selectivity of the glomerular filtration barrier in ratsfollowing laparotomy and muscle trauma. American Journal ofPhysiology—Renal Physiology, 297, F577-F582) has been added forillustration.

FIG. 3 shows a mapping of different types of blood membranes based onthe molecular weight retention onset and molecular weight cut-off fromdextran sieving curves. The dotted line squares approximately representthe boundaries that delimit the dialyzer families.

FIG. 4 depicts the effect of dialysis with high cut-off membranes onserum induced osteoblast differentiation and calcification ofmesenchymal stromal cells (MSC). MSCs were induced with osteoblastinduction medium containing serum from dialysis patients treated eitherwith conventional high-flux dialyzers (Conventional) or from the samepatients after a 3 weeks course of dialysis with high cut-off membranes(HCO). (A) Alkaline phosphatase (ALP) activity normalized to proteincontent measured on single-patient level (n-16). (B) ALP activitymeasurements from the same patients with conventional treatment set 1.0.(C) Quantification of deposited calcium normalized to protein content.Sera from a total of 16 patients were combined to 3 different serumpools for both treatment modalities. Each serum pool was applied to 4MSC preparations from different healthy donors. (D) Calcium measurementsfrom the same serum pools and cell preparations with conventionaltreatment set 1.0. ***P<0.001.

FIG. 5 depicts the dose-dependent induction of osteoblasticdifferentiation in mesenchymal stromal cells

(MSC) by pro-inflammatory cytokines, fibroblast growth factors (FGF),and full-length parathyroid hormone (PTH1-84). (A-E) Alkalinephosphatase (ALP) activity in MSCs treated with different concentrationsof IL-1β (A), TNF-α (B), FGF-2 (C), FGF-23 (D) or PTHl-84 (E) inosteoblast induction medium (OM) for 7 days. (F-J) Calcium deposited byMSCs cultured for 3 weeks in OM with increasing concentrations of IL-1β(F), TNF-α (G), FGF-2 (H), FGF-23 (I) or PTH1-84 (J). “Fold CMAX”denotes the x-fold concentration of the highest reported concentrationfound in dialysis patients (IL-1β CMAX=1.7 μg/L; TNF-α CMAX=408 ng/L;FGF-2 CMAX=19.5 ng/L; FGF-23 CMAX=255.2 ng/L; PTH1-84 CMAX=2.4 μg/L).ALP activity and calcium were normalized to sample protein content. Allvalues are expressed relative to OM without cytokine (set 1.00).Means+SEM from 4-6 independent experiments are shown. *P<0.05, **P<0.01,***P<0.001.

FIG. 6 shows the relative calcification which was measured in vitro invascular smooth muscle cells (VSMC) upon incubation with plasma samplesfrom healthy donors or from 48 patients who were dialysed with bothhigh-flux and high cut-off membranes for three weeks (Example 3.6).Calcification was assessed after 10 days with alkaline phosphatase andalizarin staining. A reduction of calcification was measured in VSMCincubated with serum after the high cut-off phase compared to thehigh-flux phase.

FIG. 7 shows the relative calcification of VSMC upon incubation withplasma samples from healthy donors and from samples obtained from invitro dialysis experiments with high cut-off, medium cut-off andhigh-flux membranes, respectively. The results of the in vitro studysupport the observation of the clinical trial (FIG. 6, Example 3.6).Vascular calcification was reduced by 36% in high cut-off probes and by32% in medium cut-off probes compared to high-flux probes.

DETAILED DESCRIPTION

Patients with impaired renal function due to chronic kidney diseasesface one of the highest risks for cardiovascular morbidity and deaththat continuously increases as kidney function declines. This is truefor patients with pre-end-stage renal failure, on dialysis or aftersuccessful renal transplantation.

The present disclosure therefore relates to a high cut-off or mediumcut-off hemodialysis membrane for the treatment of vascularcalcification in hemodialysis patients, especially in chronichemodialysis patients and especially in a CKD stage 3-5 patient havingan Agatston score of more than 11. The method comprises withdrawing andbypassing the blood from the patient in a continuous flow into contactwith one face of an hemodialysis membrane, simultaneously passingdialysate solution in a continuous flow on an opposite face of thehemodialysis membrane to the side of the hemodialysis membrane incontact with the blood, the flow of the dialysate solution beingcountercurrent to the direction of flow of blood, and returning theblood into the patient, wherein the hemodialysis membrane ischaracterized in that it comprises at least one hydrophobic polymer andat least one hydrophilic polymer and in that it has a MWRO of between 15and 20 kD and a MWCO of between 170-320 kD. Alternatively, the membranehas a MWRO of between 8.5 kD and 14.0 kD and a MWCO of between 55 and130 kD. The MWRO and MWCO for a given membrane is based on dextransieving experiments as described by Boschetti-de-Fierro et al. (2013)(see “Materials and Methods” section of the reference) and refers tovalues obtained before blood contact of the membrane.

As described before in this document, patients with CKD have adisproportionately high occurrence of vascular calcification. Onehypothesis to account for this is the altered Ca²⁺ and P²⁻ metabolismseen in these patients. Another factor mentioned is uremic toxins anduremic serum was found to upregulate the expression of, for example,Cbfa1/Runx2 and its target protein OPN, and increases secretion of amediator of osteoblastic differentiation, BMP-2, resulting in themineralization of VSMCs into osteoblast-like cells. Oxidative stress andinflammation and other inducers such as leptin are also discussed aspossible inducers. The bone proteins osteonectin, osteopontin, bonesialoprotein, type I collagen, and alkaline phosphatase have also beenidentified in multiple sites of extraskeletal calcification.Interestingly, in cell culture, vascular smooth muscle cells andvascular pericytes are capable of producing these same boneformingtranscription factors and proteins, and can be induced to do so withhigh concentrations of phosphorus, uremic serum, high glucose, oxidizedlipids, and several other factors (Moe et al. (2008): “Mechanisms ofVascular Calcification in Chronic Kidney Disease”, J Am Soc Nephrol 19,213-216.

As cells of mesenchymal origin including endothelial and vascular smoothmuscle cells are prime targets of uremic solutes, mesenchymal stromalcells (MSCs) as common progenitors of both are a suitable model toidentify mechanisms by which the uremic milieu interior may disturbvascular health. The model can also be used to determine the effect ofthe use of high cut-off membranes/dialyzers on vascular calcificationindicators in the uremic retention solutes. For the present invention,effects of 64 individual uremic retention solutes (URS) on osteoblastictransformation of MSCs were systematically studied in order to identifytherapeutic strategies feasible for targeting of vascular calcification.

Bone marrow derived MSCs were separately treated with 64 individual URSat uremic concentrations in osteoblastic induction medium. Osteoblasticdifferentiation was measured by alkaline phosphatase activity, westernblot, immunocytochemistry, and calcium deposition. In an additionaltranslational approach, osteoblastic potential of serum obtained uponhigh cut-off dialyzer treatment was compared to that obtained uponconventional dialysis. It was found in said approach that substanceremoval with high cut-off dialyzers had favorable effects on theattenuation of osteoblastic differentiation and calcium deposition byMSCs. The findings emphasize importance of larger molecules, sometimesalso referred to as “middle molecules”, in mediating uremic calcifyingMSC phenotype. Since conventional dialysis strategies fail toeffectively remove this group of URS, targeted dialysis modalities,possibly in combination with specific pharmacologic interventions werefound to be useful for addressing the unsolved problem of vascularcalcification in chronic kidney diseases.

In the present invention, the question was addressed, if maintenancedialysis with membranes characterized by a higher molecular weightcut-off and consequently a greater capacity for the removal of largermolecules compared to conventional high flux dialyzers could improveserum composition and reduce its pro-osteoblastic and pro-calcifyingeffect on MSCs. Serum from patients that had been dialyzed with highcut-off membranes for 3 weeks with serum from the same patients obtainedduring a period when they had been dialyzed with conventional high-fluxmembranes (Example 3). Overall, the potential for induction ofosteoblastic differentiation in MSCs was reduced when serum was obtainedduring HCO dialysis compared to conventional high-flux dialysis asindicated by alkaline phosphatase (ALP) activity (48.67±1.6 U/g proteinversus 65.02±4.2 U/g protein; FIG. 4A). This effect was detectable inthe paired serum samples of each patient (FIG. 4B). Finally, MSCstreated with serum from patients treated with high cut-off membranesdeposited 40% less calcium compared to serum obtained during a period ofconventional dialysis (FIG. 4C). Reduced calcification was consistentlypresent in each single experiment (FIG. 4D).

The concentrations of certain molecules found in a dialysis patient andwhich are deemed to play a role in the mediation of vascularcalcification are sometimes extremely high and will not be encounteredfrequently in stable patients on maintenance dialysis. Therefore, inorder to get an insight on dose effects of certain mediators on thedevelopment of calcification, it was tested whether or not also lowerconcentrations of inducers of MSC osteoblast differentiation havedetectable effects at least in the present in vitro model (Example 4).Dose-dependent increases were identified in both, ALP activity andcalcium deposition induced by the pro-inflammatory cytokines IL-1β(FIGS. 5, A and E) and TNF-α (FIGS. 5, B and F). The dose-response curvefor FGF-2 also revealed induction of osteoblastic differentiation andcalcium deposition at concentrations below C_(MAX) (FIGS. 5, C and G).It is obvious from these results that high cut-off membranes accordingto the invention that high cut-off dialyzers will have an effect notonly in high risk patients or those patients who already suffer fromsevere vascular calcification, but also on patients who have not yetdeveloped a severe vascular calcification and/or do not show high levelsof mediators suspected of inducing vascular calcification. In the lattercase, the onset and development of vascular calcification can beprevented or delayed.

As can be seen from FIG. 2, the high cut-off dialysis membrane allowsfor the limited passage, in whole blood, of molecules with a molecularweight of above 60 kD, including also, to a certain limited extend,albumin with a molecular weight of 68 kD. High-flux membranes, incontrast, allow only for the passage of molecules up to 25kD in wholeblood. For this reason, filters based on and comprising high cut-offmembranes can be efficiently used to remove larger molecules in therange of between 25 and 60 kD, which cannot be efficiently addressedwith conventional dialysis based on low flux or high flux dialyzer.

It was thus found, in the present invention, that in hemodialysispatients with CKD stages 3-5 and with an Agatston score of 11 and more,the use of high cut-off or medium cut-off membranes leads to a reductionof mediators inducing and/or governing vascular calcification in CKDpatients. Said use thus leads to an effective treatment of patientssuffering from vascular calcification and/or to an improved, preventivetreatment of patients having a moderate to high risk of developingvascular calcification and related cardiovascular diseases,respectively.

The use of high cut-off or medium cut-off membranes for treatinghemodialysis patients was found to be especially favorable for patientswith CKD stages 3-5 and with an

Agatston score of >100 to 400. The treatment according to the inventionis especially indicated for patients with CKD stages 3-5 and with anAgatston score of >400. The use of the high cut-off or medium cut-offmembrane in the dialysis treatment of patients of Agatston scores of11-100, 100-400 and >400 is also very advisable for patients with CKDstages 4-5. The use according to the invention of high cut-off or mediumcut-off membranes in the treatment of hemodialysis patients isespecially indicated for patients with CKD stage 5 and an Agatston scoreof >100.

The expression “vascular calcification” as used herein refers to theprocess of dedifferentiation or transformation of vascular smooth musclecells (VSMC) into osteo/chondrocytic-like cells, whereuponosteo/chondrocytic-like VSMC become calcified in a process similar tobone formation. The calcification involves deposition of collagen andnoncollagenous proteins in the intima or media and incorporation ofcalcium and phosphorus into matrix vesicles to initiate mineralizationand further mineralization into hydroxyapatite. The expression “vascularcalcification” thus encompasses, on a clinical level, arterialstiffening, higher pulse wave velocity (PWV), earlier return of wavereflections from the periphery to the ascending aorta during systole andsignificant increase of aortic systolic blood pressure with reduceddiastolic blood pressure and high pulse pressure. The vascularcalcification is quantitatively described by determining the CoronaryArtery Calcification Score (CACS) as detectable by electron-beam ormultislice computed tomography (CT) and as described before. For theavoidance of doubt, the expression “Agatston score” as used herein isequivalent to the expression Coronary Artery Calcification Score (CACS)as used herein.

In the context of the present invention, the expression “CKD patients”refers to patients with CKD (KDOQI) stages 3-5, if not indicatedotherwise. Stage 3 refers to moderately reduced kidney function with GFR(Glomerular Filtration Rate, normalized to an average surface area(size) of 1.73 m²) values of 45-59 (3A) and 30-44 (3B). Stage 4 refersto severely reduced kidney function with GFR values of 15-29. Stage 5refers to very severe or endstage kidney failure and GFR values below15.

The expression “high cut-off membrane” or “high cut-off membranes” asused herein refers to membranes comprising at least one hydrophobicpolymer and at least one hydrophilic polymer and having a MWRO ofbetween 15 and 20 kD and a MWCO of between 170-320 kD. The membranes canalso be characterized by a pore radius, on the selective layer surfaceof the membrane, of between 8-12 nm. The expression “medium cut-offmembrane” as used herein refers to membranes comprising at least onehydrophobic polymer and at least one hydrophilic polymer and having aMWRO of between 8.5 and 14.0 kD and a MWCO of between 55 kD and 130 kD.The membranes can also be characterized by a pore radius, on theselective layer surface of the membrane, of above 5.5 nm and below 8.0nm. For the avoidance of doubt, the determination of MWRO and MWCO for agiven membrane is according to the methods of Boschetti-de-Fierro et al.(2013); see “Materials and Methods” section of the reference. Theexpression “high cut-off membrane” as used herein otherwise comprisesmembranes characterized by the performance parameters as shown in TableI of this document, without wanting to limit the definition of highcut-off membranes to the single performance parameters disclosed inTable I for said membranes. The high cut-off or medium cut-off membranescan be processed into hemodialysis filters by methods generally known inthe art, for example, into hemodialysis filters having a design in termsof housing, area, fiber and bundle geometry, packing density and flowcharacteristics, similar to or the same as products already available onthe market such as, for example, HCO1100® or Theralite®, both comprisinghigh cut-off membranes. Accordingly, the use of the expression “highcut-off membrane” or “medium cut-off membrane” in the context of thepresent invention encompasses the use of the membrane within an adequatefilter device fit for being used in/on an extracorporeal dialysismachine.

In one embodiment of the invention, the high cut-off membranes for thetreatment of vascular calcification are characterized by a pore radius,on the selective surface layer of the membrane, of between 8-12 nm.

In a further embodiment of the invention, the high cut-off dialysismembrane is characterized by a clearance (ml/min) for κ-FLC of from 35to 40, and for κ-FLC of from 30 to 40 as determined according to themethod described in Table I.

In yet another embodiment of the invention, the high cut-off dialysismembranes for the treatment of vascular calcification are characterizedby allowing the passage of molecules having a molecular weight of up to45 kDa with a sieving coefficient of from 0.1 to 1.0 in presence ofwhole blood, based on EN1238 with Q_(B) max and UF 20%.

In yet another embodiment of the invention, the high cut-off dialysismembrane is characterized by sieving coefficients of from 0.9 to 1.0 forβ₂-microglobulin and of from 0.8 to 1.0 for myoglobin, when measuredaccording to EN 1283 with Q_(B) max and UF 20%.

In yet another embodiment of the invention, the medium cut-off dialysismembrane is characterized as set forth in European Patent ApplicationNo. 14154175.5.

It is a further object of the present invention to provide for a methodfor reducing and/or preventing vascular calcification in hemodialysispatients having an Agatston score of more than 11, comprisingwithdrawing and bypassing the blood from the patient in a continuousflow into contact with one face of an hemodialysis membrane,simultaneously passing dialysate solution in a continuous flow on anopposite face of the hemodialysis membrane to the side of thehemodialysis membrane in contact with the blood, the flow of thedialysate solution being countercurrent to the direction of flow ofblood, and returning the blood into the patient, wherein thehemodialysis membrane is characterized in that it comprises at least onehydrophobic polymer and at least one hydrophilic polymer and has a MWROof between 15 and 20 kD and a MWCO of between 170-320 kD, or that itcomprises at least one hydrophobic polymer and at least one hydrophilicpolymer and has a MWRO of between 8.5 and 14 kD and a MWCO of between 55kD and 130 kD

It is a further aspect of the present invention to provide for a methodfor reducing and/or preventing vascular calcification in hemodialysispatients having an Agatston score of more than 100. It is another aspectof the present invention to provide for a method for reducing and/orpreventing vascular calcification in hemodialysis patients having anAgatston score of between 100 and 400. It is yet a another aspect of thepresent invention to provide for a method for reducing and/or preventingvascular calcification in hemodialysis patients having an Agatston scoreof more than 400. It is also an aspect of the present invention toprovide for a method for reducing and/or preventing vascularcalcification in hemodialysis patients having an Agatston score of morethan 11 and with CKD stages 3-5, especially 4-5. It is a further aspectof the present invention to provide for a method for reducing and/orpreventing vascular calcification in hemodialysis patients having anAgatston score of more than 100 and with CKD stages 4-5, especially 5.

It is another aspect of the present invention to provide for a dialysismembrane comprising at least one hydrophobic polymer and at least onehydrophilic polymer, wherein the membrane allows the passage ofmolecules having a molecular weight of up to 45 kDa with a sievingcoefficient of from 0.1 to 1.0 in presence of whole blood, based onEN1238 with Q_(B) max and UF 20%, for treating vascular calcification ina hemodialysis patient, especially in hemodialysis patients with anAgatston score of >11.

It is also an aspect of the present invention to provide for a dialysismembrane comprising at least one hydrophobic polymer and at least onehydrophilic polymer, wherein the membrane has a molecular weightretention onset (MWRO) of between 15 and 20 kD and a MWCO of between170-320 kD for treating vascular calcification in hemodialysis patients,especially in hemodialysis patients with an Agatston score of >11,wherein the membrane has a pore radius, on the selective layer, ofbetween 8 and 12 nm.

It is also an aspect of the present invention to provide for a dialysismembrane comprising at least one hydrophobic polymer and at least onehydrophilic polymer, wherein the membrane has a molecular weightretention onset (MWRO) of between 8.5 kD and 14 kD and a MWCO of between55 kD and 130 kD for treating vascular calcification in hemodialysispatients, especially in hemodialysis patients with an Agatston scoreof >11, wherein the membrane has a pore radius, on the selective layer,of between 8 and 12 nm.

In another embodiment of the invention, the hemodialysis treatmentregime is performed with a high cut-off or medium cut-off membrane whichhas a urea clearance of at least 170 ml/min at a Q_(B) of 200 ml/min anda Q_(D) of 500 ml/min (UF=0 ml/min). In yet another embodiment of theinvention, the dialysis treatment according to the invention must ensurea Kt/V of >1.2.

In yet another embodiment of the invention, a patient's total albuminloss does not exceed about 60 g per week, and preferably does not exceed40 g per week.

In one embodiment of the invention, the hemodialysis treatment with themembranes according to the invention is performed from 2 to 4 times perweek for a period of from 2 to 6 hours, respectively, with a membraneaccording to the invention. A hemodialysis patient suffering fromvascular calcification, especially a CKD patient with stage 3-5, is thusbeing treated, for a certain period of time, only with such hemodialysisfilter according to the invention. In one embodiment of the invention,the treatment may continue until the signs of vascular calcificationhave been stayed or have decreased. In another embodiment of theinvention, the patient receives a continual standard hemodialysistreatment with a hemodialysis filter comprising a medium cut-offmembrane. In the context of the present invention, “stayed” and/or“decreased” refers to a constant Agatston score or the reduction of theAgatston score, respectively. According to another embodiment of theinvention, the treatment regimen as described may be applied for aperiod of from 4 to 12 weeks. In yet another embodiment of theinvention, the treatment may continually be used for a hemodialysispatient with stage 3-5, especially a patient who belongs to a medium tohigh or high risk group as defined by the Agatston score.

In another embodiment of the invention, one of three hemodialysistreatments per week is performed for a period of 2 to 6 hours with amembrane according to the invention, whereas two of three hemodialysistreatments per week comprise the use of a standard high-fluxhemodialysis membrane. Said treatment may be used in cases wherestandard dialysis is recommended in addition to using a hemodialysisfilter according to the invention. In one embodiment of the invention,the treatment may continue until the signs of vascular calcificationhave been stayed or have decreased, or until an Agatston score of below100, preferably below 50 has been reached. In another embodiment of theinvention, the treatment regime as described may be applied for a periodof from 4 to 12 weeks. In yet another embodiment of the invention, thetreatment may continually be used for a hemodialysis patient with CDKstage 3-5, especially a patient who belongs to a medium to high or highrisk group as defined by the Agatston score.

In a further embodiment of the present invention, the hemodialysistreatment for a period of 2 to 6 hours is performed with a dialysisfilter comprising a membrane according to the invention every otherdialysis treatment, whereas the other hemodialysis treatment comprisesthe use of a standard high-flux hemodialysis membrane. Said treatmentmay be used in cases where standard dialysis is recommended in additionto using a hemodialysis filter according to the invention. In oneembodiment of the invention, the treatment may continue until the signsof vascular calcification have been stayed or have decreased, or untilan Agatston score of below 100, preferably below 50 has been reached.

Depending on the specific condition of a patient, such treatmentregimens or routines can be applied singularly or dynamically, i.e. theymay be interchanged or subsequently be used for certain periods of time.

Accordingly, the above method also provides for a possibility to reduceor suspend the further development of vascular calcification inhemodialysis patients. The treatment according to the invention isdesigned to reduce or remove such molecules which are connected to thecondition of vascular calcification as discussed before. Theamelioration of the condition of the patient based on the presenttreatment will allow reducing medication which has to be administered tothe patients and the risk going hand in hand with such medication asdescribed before. The respective reduction rates upon using a highcut-off or medium cut-off membrane according to the invention at leastlie in the range of more than 10% relative to the Agatston scoredetermined at the beginning of treating a given patient according to theinvention. It is an object of the present invention to achieve reductionrates of more than 20%, preferably more than 30%. At least the use ofhigh cut-off or medium cut-off membranes and filter devices comprisingthem is connected to no further increase of the Agatston scoredetermined at the beginning of treating a given patient according to theinvention.

In one embodiment of the invention, the hemodialysis treatment accordingto the invention can be supplemented by a state of the art medicationwhich would otherwise be prescribed to a patient suffering from vascularcalcification.

Dialysis machines which can be used for performing a treatment accordingto the invention are standard dialysis machines which can accuratelycontrol and monitor the ultrafiltration rate. Examples for such devicesare the AK 96™, AK 200™ S and AK 200™ ULTRA S, PrismafleX eXeed™ or theArtis™ dialysis machines of Gambro Lundia AB. However, any otherdialysis machine having UF control can also be used for the treatment.

Parameters for performing a treatment according to the invention can beadjusted to standard dialysis treatment or medium cut-off parameters andthe specifications of the high cut-off or medium cut-off membrane.Typical flow rates used for the present treatment may vary. It isadvantageous to use flow rates with a Q_(B) (blood flow) of 100-500,preferably 250-400 ml/min and a Q_(D) (dialysate flow rate) of 100-1000,preferably 300-500 ml/min.

Membrane passage of a solute, such as a protein which needs to beremoved from blood, is described by means of the sieving coefficient S.The sieving coefficient S is calculated according toS=(2C_(F))/(C_(Bin)+C_(Bout)), where C_(F) is the concentration of thesolute in the filtrate and C_(Bin) is the concentration of a solute atthe blood inlet side of the device under test, and C_(Bout) is theconcentration of a solute at the blood outlet side of the device undertest. A sieving coefficient of S=1 indicates unrestricted transportwhile there is no transport at all at S=0. For a given membrane eachsolute has its specific sieving coefficient.

In addition, the sieving curves may serve as a basis for determining,for example, the average or mean pore size or pore size distribution ofa membrane on the selective layer. There is a factual and mathematicalcorrelation between the sieving characteristics of a membrane and itspore structure. The mean pore size or pore size distribution can, forexample, be determined according to Aimar et al (1990) from the dextransieving curve.

In one embodiment, the membrane allows for the passage of free lightchains (FLC). That is, the κ or λ free light chains pass through themembrane. High flux membranes, with smaller pore sizes, sometimes alsoreferred to as “protein-leaking membranes”, have been observed to removesome free light chains. However, this appears to be primarily due tobinding of the FLC onto the dialysis membranes. FLC may be used asmarkers of middle molecular weight proteins. Although clearing of freelight chains is not a primary target of the invention, their reductioncan be used as an indicator of membrane functionality.

It is provided, in a further aspect of the invention, dialysis systemwherein the membrane has a clearance (ml/min) for κ-FLC of from 30 to45, and for λ-FLC of from 28 to 40. Clearance is determined in vitro(±20%) with Q_(B)=250 ml/min, Q_(D)=500 ml/min, UF=0 ml/min in bovineplasma having a protein level of 60 g/l at 37° C. The plasma level forhuman κ=500 mg/l and for human λ=250 mg/l.

In one aspect of the present invention, the dialysis membrane accordingto the invention comprises at least one hydrophilic polymer and at leastone hydrophobic polymer. In one embodiment, at least one hydrophilicpolymer and at least one hydrophobic polymer are present in the dialysismembrane as domains on the surface of the dialysis membrane.

The hydrophobic polymer may be chosen from the group consisting ofpolyarylethersulfone (PAES), polypropylene (PP), polysulfone (PSU),polymethylmethacrylate (PMMA), polycarbonate (PC), polyacrylonitrile(PAN), polyamide (PA), polytetrafluorethylene (PTFE) or combinationsthereof. In one embodiment of the invention, the hydrophobic polymer ischosen from the group consisting of polyarylethersulfone (PAES),polypropylene (PP), polysulfone (PSU), polycarbonate (PC),polyacrylonitrile (PAN), polyamide (PA) polytetrafluorethylene (PTFE) orcombinations thereof. In another embodiment of the invention, thehydrophobic polymer is chosen from the group consisting ofpolyarylethersulfone (PAES) and polysulfone (PSU).

The hydrophilic polymer may be chosen from the group consisting ofpolyvinylpyrrolidone (PVP), polyethyleneglycol (PEG), polyvinylalcohol(PVA), and copolymer of polypropyleneoxide and polyethyleneoxide(PPO-PEO). In one embodiment of the invention, the hydrophilic polymermay be chosen from the group consisting of polyvinylpyrrolidone (PVP),polyethyleneglycol (PEG) and polyvinylalcohol (PVA). In one embodimentof the invention, the hydrophilic polymer is polyvinylpyrrolidone (PVP).

In one embodiment of the invention, the high cut-off dialysis membraneis a hollow fiber having a symmetric (sponge-like) or an asymmetricstructure with a separation layer present in the innermost layer of thehollow fiber. In one embodiment of the invention, the high cut-offdialysis membrane has at least a 3-layer asymmetric structure, whereinthe separation layer has a thickness of less than 0.5 μm. In oneembodiment, the separation layer contains pore channels having anaverage pore size of more than 7 nm, generally between 8 and 12 nm asbased on dextran sieving coefficients (see also Boschetti-de-Fierreo etal. (2013), Table III). The average pore size (diameter) is generallyabove 8 nm for this type of membrane (FIG. 6). The next layer in thehollow fiber membrane is the second layer, having the form of a spongestructure and serving as a support for said first layer. In a preferredembodiment, the second layer has a thickness of about 1 to 15 μm. Thethird layer has the form of a finger structure. Like a framework, itprovides mechanical stability on the one hand; on the other hand a verylow resistance to the transport of molecules through the membrane, dueto the high volume of voids. During the transport process, the voids arefilled with water and the water gives a lower resistance againstdiffusion and convection than a matrix with a sponge-filled structurehaving a lower void volume. Accordingly, the third layer providesmechanical stability to the membrane and, in a preferred embodiment, hasa thickness of 20 to 60 μm.

In one embodiment, the high cut-off dialysis membrane also includes afourth layer, which is the outer surface of the hollow fiber membrane.In this embodiment, the outer surface has openings of pores in the rangeof 0.5 to 3 μm and the number of said pores is in the range of from10.000 to 150.000 pores/mm², preferably 20.000 to 100.000 pores/mm².This fourth layer preferably has a thickness of 1 to 10 μm.

The manufacturing of a high cut-off dialysis membrane follows a phaseinversion process, wherein a polymer or a mixture of polymers isdissolved in a solvent to form a polymer solution. The solution isdegassed and filtered and is thereafter kept at an elevated temperature.Subsequently, the polymer solution is extruded through a spinning nozzle(for hollow fibers) or a slit nozzle (for a flat film) into a fluid bathcontaining a non-solvent for the polymer. The non-solvent replaces thesolvent and thus the polymer is precipitated to an inverted solid phase.

To prepare a hollow fiber membrane, the polymer solution preferably isextruded through an outer ring slit of a nozzle having two concentricopenings. Simultaneously, a center fluid is extruded through an inneropening of the nozzle. At the outlet of the spinning nozzle, the centerfluid comes in contact with the polymer solution and at this time theprecipitation is initialized. The precipitation process is an exchangeof the solvent from the polymer solution with the non-solvent of thecenter fluid.

By means of this exchange the polymer solution inverses its phase fromthe fluid into a solid phase. In the solid phase the pore structure,i.e. asymmetry and the pore size distribution, is generated by thekinetics of the solvent/non-solvent exchange. The process works at acertain temperature which influences the viscosity of the polymersolution. The temperature at the spinning nozzle and the temperature ofthe polymer solution and center fluid is 30 to 80° C. The viscositydetermines the kinetics of the pore-forming process through the exchangeof solvent with non-solvent. The temperature in the given range shouldbe chosen in way to be some degrees higher than the temperature whichwould have been chosen for the same recipe in order to obtain a standardhigh-flux membrane. Subsequently, the membrane is preferably washed anddried.

By the selection of precipitation conditions, e. g. temperature andspeed, the hydrophobic and hydrophilic polymers are “frozen” in such away that a certain amount of hydrophilic end groups are located at thesurface of the pores and create hydrophilic domains. The hydrophobicpolymer builds other domains. A certain amount of hydrophilic domains atthe pore surface area are needed to avoid adsorption of proteins. Thesize of the hydrophilic domains should preferably be within the range of20 to 50 nm. In order to repel albumin from the membrane surface, thehydrophilic domains also need to be within a certain distance from eachother. By the repulsion of albumin from the membrane surface, directcontact of albumin with the hydrophobic polymer, and consequently theabsorption of albumin, are avoided.

The polymer solution used for preparing the membrane preferablycomprises 10 to 20 wt.-% of hydrophobic polymer and 2 to 11 wt.-% ofhydrophilic polymer. The center fluid generally comprises 45 to 60 wt.-%of precipitation medium, chosen from water, glycerol and other alcohols,and 40 to 55 wt.-% of solvent. In other words, the center fluid does notcomprise any hydrophilic polymer.

In one embodiment, the polymer solution coming out through the outerslit openings is, on the outside of the precipitating fiber, exposed toa humid steam/air mixture. Preferably, the humid steam/air mixture has atemperature of at least 15° C., more preferably at least 30° C., and notmore than 75° C., more preferably not more than 60° C.

Preferably, the relative humidity in the humid steam/air mixture isbetween 60 and 100%. Furthermore, the humid steam in the outeratmosphere surrounding the polymer solution emerging through the outerslit openings preferably includes a solvent. The solvent content in thehumid steam/air mixture is preferably between 0.5 and 5.0 wt-%, relatedto the water content. The effect of the solvent in thetemperature-controlled steam atmosphere is to control the speed ofprecipitation of the fibers. When less solvent is employed, the outersurface will obtain a denser surface, and when more solvent is used, theouter surface will have a more open structure. By controlling the amountof solvent within the temperature-controlled steam atmospheresurrounding the precipitating membrane, the amount and size of the poreson the outer surface of the membrane are controlled, i.e. the size ofthe openings of the pores is in the range of from 0.5 to 3 μm and thenumber of said pores is in the range of from 10,000 to 150,000pores/mm². A fourth layer of a high cut-off dialysis membrane ispreferably prepared by this method.

Before the extrusion, suitable additives may be added to the polymersolution. The additives are used to form a proper pore structure andoptimize the membrane permeability, the hydraulic and diffusivepermeability, and the sieving properties. In a preferred embodiment, thepolymer solution contains 0.5 to 7.5 wt.-% of a suitable additive,preferably chosen from the group comprising water, glycerol and otheralcohols.

The solvent may be chosen from the group comprising N-methylpyrrolidone(NMP), dimethyl acetamide (DMAC), dimethyl sulfoxide (DMSO) dimethylformamide (DMF), butyrolactone and mixtures of said solvents.

Medium cut-off membranes can be prepared as described in EP 14154175.5.

Membranes which can also effectively be used according to the inventionand methods for preparing them are also described in EP 2 253 367 A1.Dialysis filters which can be used according to the invention are shown,for example, in Table II of Boschetti-de-Fierro et al (2013) andidentified as “High cut-off” dialyzer.

It will be readily apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

The present invention will now be illustrated by way of non-limitingexamples in order to further facilitate the understanding of theinvention.

EXAMPLES Example 1 High Cut-Off Membrane Preparation

Two solutions are used for the formation of the membrane, the polymersolution consisting of hydrophobic and hydrophilic polymer components(21 wt-%) dissolved in N-methyl-pyrrolidone, and the center solutionbeing a mixture of N-methyl-pyrrolidone and water. The polymer solutioncontains polyethersulfone (PES 14.0 wt-%) and polyvinylpyrrolidone (PVP7.0 wt-%) as membrane building components. The solution further containsNMP (77.0 wt-%) and water (2.0 wt-%). The center solution contains water(53.0 wt-%) and NMP (47.0 wt-%).

During the membrane formation process polymer and center solution arebrought in contact with a spinneret or jet and the membraneprecipitates. A defined and constant temperature (58° C.) is used tosupport the process. The precipitated hollow fiber falls through ahumidified shaft filled with steam (100% relative humidity, 54° C.) intoa washing bath (20° C., ˜4 wt-% NMP). The membrane is further washed intwo additional water baths (70° C.-90° C.) with counter current flow(250 l/h). Membrane drying is performed online, wherein remaining wateris removed.

Fibers used in the following tests had an inner diameter of 215 μm, awall thickness of 50 μm, and an effective membrane area of, for example1.1 m² (as in HCO1100®) or 2.1 m² (as in Theralite®).

Example 2 Preparation of Hand Bundles, Mini-Modules and Filters

The preparation of a membrane bundle after the spinning process isnecessary to prepare the fiber bundle for following performance testswith mini-modules. The first process step is to cut the fiber bundles toa defined length of 23 cm. The next process step consists of melting theends of the fibers. An optical control ensures that all fibers are wellmelted. Then, the ends of the fiber bundle are transferred into apotting cap. The potting cap is fixed mechanically and a potting tube isput over the potting caps. Then the fibers are potted with polyurethane.After the polyurethane has hardened, the potted membrane bundle is cutto a defined length and stored dry before it is used for the differentperformance tests.

Mini-modules [=fiber bundles in a housing] are prepared in a similarmanner. The mini-modules ensure protection of the fibers and are usedfor steam-sterilization. The manufacturing of the mini-modules comprisesthe following specific steps:

-   (A) The number of fibers required is calculated for an effective    surface A of 360 cm² according to equation (1)

A=π×d _(i) ×l×n[cm²]  (1)

-   -   Wherein d_(i) is the inner diameter of fiber [cm], n represents        the amount of fibers, and l represents the effective fiber        length [cm].

-   (B) The fiber bundle is cut to a defined length of 20 cm.

-   (C) The fiber bundle is transferred into the housing before the    melting process

-   (D) The mini-module is put into a vacuum drying oven over night    before the potting process.

For in vivo studies standard format dialysis filters are needed. Suchfilters can be prepared from the hollow fiber membranes of Example 1according to methods known in the art. Fiber geometry is as said beforein Example 1. The blood flow range can be from 100-400 ml/min. Forexample, the HCO1100® dialyzer has a blood flow range of 200-500 ml/min,the Theralite® dialyzer has a blood flow range of 100 to 400 ml/min.Dialysate flow range is from 300 to 800 ml/min. For example, theHCO1100® dialyzer has a dialysate flow range of 300-800 ml/min, theTheralite® dialyzer has a dialysate flow range of up to 800 ml/min.

Example 3

Effects of Dialysis with High Cut-Off Membranes on the Ability of Serumto Induce Osteoblastic Differentiation in MSCs

The study was conducted in accordance with the Declaration of Helsinkiand had been approved by local ethic authorities. All subjects providedwritten informed consent.

3.1 Isolation and Culture of MSCs

MSCs were isolated from bone marrow aspirates obtained from 20 healthybone marrow donors (7 female, 13 male) median age 31 years (range0.5-42) as described previously (Lange et al. (2007), J Cell Physiol213, 18-26). All subjects provided written informed consent. In brief,bone marrow mononuclear cells were purified by Percoll density gradientcentrifugation, plated at 400,000 cells/cm² and cultured in α-MEM(#E15-862, PAA) supplemented with 100 U/mL penicillin (PAA), 100 μg/mLstreptomycin (PAA), 2 IU/ml heparin (Ratiopharm), and 5% freshly thawedplatelet lysate at 37° C. and 5% CO₂. Nonadherent cells were washed offwith PBS after 2-3 days. Medium was changed twice a week. When culturesreached about 90% confluence, cells were detached with 0.05%Trypsin/0.02% EDTA (PAA), counted, and re-plated at 500 cells/cm² in 175cm² flasks (Saarstedt). For all MSC preparations, mesenchymalmultilineage differentiation capacity, expression of characteristicsurface marker proteins (CD59, CD90, CD105), and lack of hematopoeticmarkers were confirmed (supplemental Figure S1) according to thestandard criteria for MSC research.

3.2 Induction of Osteoblastic Differentiation

Passages 2 to 5 were used for experiments. MSCs were seeded in completea-MEM at 141,000 cells per well in 6-well-plates. The next day, mediumwas changed to osteoblast induction medium (OM) consisting of Dulbecco'sModified Eagle's Medium (DMEM; PAA) supplemented with 2 mM glutamine(PAA), penicillin/streptomycin (PAA), 1% FCS (Biochrome), 10 mMβ-glycerophosphate, 500 μM ascorbic acid, and 100 nM dexamethasone (allfrom Sigma).

3.3 High Cut-Off Versus Conventional Dialysis Membranes

For the assessment of enhanced removal of relevant mediators of vascularcalcification by dialysis and the effects on MSC osteoblasticdifferentiation, serum from 16 dialysis patients treated with eitherconventional (Polyflux 210H, Gambro) or high cut-off membranes accordingto the invention (HCO1100®, in line with a Polyflux® 14L dialyzer forreaching a sufficient Kt/V due to the limited membrane area of theHCO1100® dialyzer) were tested. Serum was obtained immediately before adialysis session after a dialysis-free interval of 3 days. One serumsample was taken after at least 3 weeks of dialysis treatment with theconventional high-flux (HFL) membrane. Another serum sample was drawnfrom the same patients after they had been dialyzed for 3 weeks withhigh cut-off membranes. One half of the patients were treated with theHFL dialyzer prior to the high cut-off dialyzer. The other half receivedthe different treatments in opposite sequence. OM was supplemented with2.5% patient serum instead of 1% FCS. Medium was changed every 2-3 days.

3.4 Alkaline Phosphatase Activity

Activity of alkaline phosphatase (ALP) in MSCs was determined afterexposure to the different experimental conditions for 7 days. Cells werewashed with PBS and lysed with 400 μl ALP lysis buffer (150 mM Tris pH10.0, 0.1 mM ZnCl₂, 0.1 mM MgCl₂, 1% Triton-X100) at room temperatureunder constant agitation for 30 minutes. Supernatants were collected andaliquots were immediately frozen at −80° C. For measurement of ALPactivity, an aliquot was thawed and centrifuged for 10 min at 12,000 rpmand 4° C. Each sample was measured in triplicate. 50 μl per well of a96-well-plate were mixed with 200 μl substrate solution (ALP buffer withfreshly dissolved p-Nitrophenyl phosphate at 2.7 mM) that was pre-warmedto 37° C. Optical densities (OD) were measured at 405 nm and followedevery 10 min over a 1-h incubation period at 37° C. DOD values tobaseline ODs at one chosen time point during the linear phase weredivided by the protein concentration of the sample as determined withthe DC Protein Assay (Bio-Rad). Each LOD/protein ratio was related tothe ΔOD/protein ratio of the appropriate control.

3.5 Calcium Deposition

Extracellular calcium deposition by differentiating MSCs was assessedafter 3 weeks of incubation with OM and different experimentalsubstances or earlier if the cells started dying due to extensivecalcification. After supernatants were discarded calcified cells werescraped off in 500 μL 0.6 M HCl, transferred to microtubes and incubatedovernight under constant agitation at 4° C. to solubilize the calcium.Samples were then centrifuged for 60 min at 20,000 g and 4° C.Supernatants were transferred to new microtubes and pellets weredissolved in 25 μl 0.1 M NaOH/0.1% SDS solution for proteinquantification with the DC protein assay (Bio-Rad). Supernatants wereassayed in duplicate in 96-well-plates. 10 μL either of a calciumstandard curve ranging from 5 to 25 mg/dL or sample were mixed with 150μL color reagent (0.1 mg/mL ortho-cresophthalein complexone, 1 mg/mL8-hydroxy-quinoline, 0.7 M HCl) and 150 μl AMP buffer (15%2-amino-2-methyl-1-propanol in H₂O, pH 10.7, adjusted with HCl). Afterincubation for 15 min at room temperature OD was measured at 540 nm.Blank absorption was subtracted and calcium concentrations werecalculated by means of the standard curve. Extracellular calcium wasfinally expressed as μg calcium per mg protein.

3.5 Statistics

All data are expressed as mean+SEM. The screening experiments wereevaluated with the Wilcoxon signed-rank test or, after confirming normaldistribution of the data with the Kolmogorov-Smirnov test, with thet-test. 1-way ANOVA followed by Dunnett's post-test was used to evaluatedose-response curves. The Wilcoxon matched pairs test was performed tocompare the effect of the two dialysis membranes. All analyses wereperformed with GraphPad Prism version 5.02 for Windows, GraphPadSoftware, San Diego Calif. USA. Significance was considered at a valueof p<0.05.

3.6 In Vitro Calcification of VSMC

During the study 48 patients were dialyzed with both high-flux and highcut-off membranes for three weeks. After each phase plasma serum sampleswere drawn and incubated with calcifying smooth muscle cells asdescribed before. After ten days calcification was assessed withalkaline phosphatase and alizarin staining. A reduction of calcificationwas measured in VSMC which had been incubated with serum after the highcut-off phase compared to high-flux phase (FIG. 6).

Apart from the clinical trial an in vitro dialysis model wasestablished. Briefly, plasma samples were obtained from healthy donorsand incubated with lipopolysacharide for 3 hours. Afterwards the plasmasamples were dialyzed with high cut-off, high-flux and medium cut-offmembranes in an in vitro model. The plasma samples obtained wereincubated in the cell culture model described above. The incubation ofVSMC with plasma samples from the in vitro dialysis supports theobservations of the clinical trial. Vascular calcification was reducedby 36% with high cut-off probes and by 32% with medium cut-off probescompared to high flux dialysis (FIG. 7).

1.-16. (canceled)
 17. A method of reducing induction of mesenchymalstromal cells in blood, said method comprising the step of contactingthe blood with one face of a hemodialysis membrane, simultaneouslypassing dialysate solution in a continuous flow on an opposite face ofthe hemodialysis membrane to the side of the hemodialysis membrane incontact with the blood, the flow of the dialysate solution beingcountercurrent to the direction of flow of blood, wherein thehemodialysis membrane comprises at least one hydrophobic polymer and atleast one hydrophilic polymer and has a Molecular Weight Retention Onset(MWRO) of between 15 and 20 kD and a Molecular Weight Cut-Off (MWCO) ofbetween 170-320 kD as determined by dextran sieving before blood contactof the membrane, and wherein the step of contacting the blood with thehemodialysis membrane reduces the induction of the mesenchymal stromalcells.
 18. The method of claim 1, wherein the reduction of induction isa reduced induction of osteoblastic phenotype of the mesenchymal stromalcells.
 19. The method of claim 1, wherein the reduction of induction isa reduced induction of osteoblastic differentiation of the mesenchymalstromal cells.
 20. The method of claim 1, wherein the reduction ofinduction is a reduced induction of mesenchymal stromal cellcalcification.
 21. The method of claim 1, wherein the hemodialysismembrane has a MWRO of between 8.5 kD and 14.0 kD and a MWCO of between55 kD and 130 kD as determined by dextran sieving before blood contactof the membrane.
 22. The method of claim 1, wherein the hemodialysismembrane has a pore radius, on the selective layer surface of themembrane, of between 8-12 nm.
 23. The method of claim 1, wherein thehemodialysis membrane has a pore radius, on the selective layer surfaceof the membrane, of more than 5.5 nm and less than 8.0 nm.
 24. Themethod of claim 1, wherein the hemodialysis membrane allows passage ofmolecules having a molecular weight of up to 45 kDa with a sievingcoefficient of from 0.1 to 1.0 in presence of whole blood, based onEN1238 with Q_(B) max and UF 20%.
 25. The method of claim 1, wherein themembrane has a clearance (ml/min) for κ-free light chains (κ-FLC) offrom 30 to
 45. 26. The method of claim 1, wherein the membrane has aclearance (ml/min) for λ-free light chains (λ-FLC) of from 28 to
 40. 27.The method of claim 1, wherein the blood is obtained from a hemodialysispatient.
 28. The method of claim 21, wherein the blood is obtained froma hemodialysis patient.
 29. The method of claim 22, wherein the blood isobtained from a hemodialysis patient.
 30. The method of claim 27,wherein the hemodialysis patient is classified in one of Chronic KidneyDisease (CKD) stages 3, 4 or 5 and has an Agatston score of above 10.31. The method of claim 27, wherein the hemodialysis patient isclassified in one of Chronic Kidney Disease (CKD) stages 4 or 5 and hasan Agatston score of above
 100. 32. The method of claim 27, wherein thehemodialysis patient undergoes hemodialysis treatment performed from 2to 4 times per week for a period of from 2 to 6 hours.
 33. The method ofclaim 27, wherein the hemodialysis patient has an Agatston score ofabove
 10. 34. The method of claim 27, wherein the membrane has areduction rate of the patient Agatston score of more than 10%.
 35. Themethod of claim 27, wherein the membrane has a reduction rate of thepatient Agatston score of more than 20%.
 36. The method of claim 11,wherein the membrane has a reduction rate of the patient Agatston scoreof more than 30%.